Aging drives cerebrovascular network remodeling and functional changes in the mouse brain.

Aging is frequently associated with compromised cerebrovasculature and pericytes. However, we do not know how normal aging differentially impacts vascular structure and function in different brain areas. Here we utilize mesoscale microscopy methods and in vivo imaging to determine detailed changes in aged murine cerebrovascular networks. Whole-brain vascular tracing shows an overall ~10% decrease in vascular length and branching density with ~7% increase in vascular radii in aged brains. Light sheet imaging with 3D immunolabeling reveals increased arteriole tortuosity of aged brains. Notably, vasculature and pericyte densities show selective and significant reductions in the deep cortical layers, hippocampal network, and basal forebrain areas. We find increased blood extravasation, implying compromised blood-brain barrier function in aged brains. Moreover, in vivo imaging in awake mice demonstrates reduced baseline and on-demand blood oxygenation despite relatively intact neurovascular coupling. Collectively, we uncover regional vulnerabilities of cerebrovascular network and physiological changes that can mediate cognitive decline in normal aging.

Developmental Mouse Brain Common Coordinate Framework.

3D standard reference brains serve as key resources to understand the spatial organization of the brain and promote interoperability across different studies. However, unlike the adult mouse brain, the lack of standard 3D reference atlases for developing mouse brains has hindered advancement of our understanding of brain development. Here, we present a multimodal 3D developmental common coordinate framework (DevCCF) spanning mouse embryonic day (E) 11.5, E13.5, E15.5, E18.5, and postnatal day (P) 4, P14, and P56 with anatomical segmentations defined by a developmental ontology. At each age, the DevCCF features undistorted morphologically averaged atlas templates created from Magnetic Resonance Imaging and co-registered high-resolution templates from light sheet fluorescence microscopy. Expert-curated 3D anatomical segmentations at each age adhere to an updated prosomeric model and can be explored via an interactive 3D web-visualizer. As a use case, we employed the DevCCF to unveil the emergence of GABAergic neurons in embryonic brains. Moreover, we integrated the Allen CCFv3 into the P56 template with stereotaxic coordinates and mapped spatial transcriptome cell-type data with the developmental ontology. In summary, the DevCCF is an openly accessible resource that can be used for large-scale data integration to gain a comprehensive understanding of brain development.

Comparing Left Ventricular Diastolic Function between Peritoneal Dialysis and Non-Dialysis Patients with Stage 5 Chronic Kidney Disease: A Propensity Score-Matched Analysis.

Patients with chronic kidney disease (CKD) have a high incidence of left ventricular diastolic dysfunction (LVDD), which increases the risk of heart failure and mortality. We assessed fluid overload as an independent risk factor for LVDD in patients with decreased kidney function and compared its impact on the E/e' ratio as a parameter for assessing left ventricular diastolic functions between patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and those with non-dialysis CKD stage 5 (CKD5) using propensity score matching (PSM). After PSM, 222 patients (CAPD, n = 111; CKD5, n = 111) were included. Fluid balance was assessed using bio-impedance spectroscopy and LVDD was determined by echocardiography based on an E/e' ratio of >15. The CKD5 group had a significantly higher E/e' ratio (p = 0.002), while fluid overload (OH/ECW) did not differ significantly between the groups. In the CAPD group, there were no significant differences in OH/ECW between patients with and without LVDD (p = 0.517). However, in the CKD5 group, patients with LVDD showed a significantly higher OH/ECW (p = 0.001). In a regression analysis investigating factors associated with the E/e' ratio, OH/ECW was not significantly associated with the E/e' ratio in the CAPD group (p = 0.087), but in the CKD5 group, it was independently correlated (p = 0.047). The factors closely associated with LVDD varied depending on dialysis dependence. While fluid overload independently influenced LVDD in non-dialysis patients, it was not statistically significant in patients with CAPD. Early assessment and management of volume status are crucial in addressing LVDD in patients with advanced-stage CKD.

MIG-6 is essential for promoting glucose metabolic reprogramming and tumor growth in triple-negative breast cancer.

Treatment of triple-negative breast cancer (TNBC) remains challenging due to a lack of effective targeted therapies. Dysregulated glucose uptake and metabolism are essential for TNBC growth. Identifying the molecular drivers and mechanisms underlying the metabolic vulnerability of TNBC is key to exploiting dysregulated cancer metabolism for therapeutic applications. Mitogen-inducible gene-6 (MIG-6) has long been thought of as a feedback inhibitor that targets activated EGFR and suppresses the growth of tumors driven by constitutive activated mutant EGFR. Here, our bioinformatics and histological analyses uncover that MIG-6 is upregulated in TNBC and that MIG-6 upregulation is positively correlated with poorer clinical outcomes in TNBC. Metabolic arrays and functional assays reveal that MIG-6 drives glucose metabolism reprogramming toward glycolysis. Mechanistically, MIG-6 recruits HAUSP deubiquitinase for stabilizing HIF1α protein expression and the subsequent upregulation of GLUT1 and other HIF1α-regulated glycolytic genes, substantiating the comprehensive regulation of MIG-6 in glucose metabolism. Moreover, our mouse studies demonstrate that MIG-6 regulates GLUT1 expression in tumors and subsequent tumor growth in vivo. Collectively, this work reveals that MIG-6 is a novel prognosis biomarker, metabolism regulator, and molecular driver of TNBC.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

Mitochondrial Superoxide Dismutase Has a Protumorigenic Role in Ovarian Clear Cell Carcinoma.

Epithelial ovarian cancer (EOC) is the fourth leading cause of death due to cancer in women and comprises distinct histologic subtypes, which vary widely in their genetic profiles and tissues of origin. It is therefore imperative to understand the etiology of these distinct diseases. Ovarian clear cell carcinoma (OCCC), a very aggressive subtype, comprises >10% of EOCs. In the present study, we show that mitochondrial superoxide dismutase (Sod2) is highly expressed in OCCC compared with other EOC subtypes. Sod2 is an antioxidant enzyme that converts highly reactive superoxide (O2 (•-)) to hydrogen peroxide (H2O2) and oxygen (O2), and our data demonstrate that Sod2 is protumorigenic and prometastatic in OCCC. Inhibiting Sod2 expression reduces OCCC ES-2 cell tumor growth and metastasis in a chorioallantoic membrane (CAM) model. Similarly, cell proliferation, migration, spheroid attachment and outgrowth on collagen, and Akt phosphorylation are significantly decreased with reduced expression of Sod2. Mechanistically, we show that Sod2 has a dual function in supporting OCCC tumorigenicity and metastatic spread. First, Sod2 maintains highly functional mitochondria, by scavenging O2 (•-), to support the high metabolic activity of OCCC. Second, Sod2 alters the steady-state ROS balance to drive H2O2-mediated migration. While this higher steady-state H2O2 drives prometastatic behavior, it also presents a doubled-edged sword for OCCC, as it pushed the intracellular H2O2 threshold to enable more rapid killing by exogenous sources of H2O2. Understanding the complex interaction of antioxidants and ROS may provide novel therapeutic strategies to pursue for the treatment of this histologic EOC subtype.

Overexpression of Id1 in transgenic mice promotes mammary basal stem cell activity and breast tumorigenesis.

Inhibitor of differentiation/DNA binding (Id)1 is a crucial regulator of mammary development and breast cancer progression. However, its effect on stemness and tumorigenesis in mammary epithelial cells remains undefined. Herein, we demonstrate that Id1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities in transgenic mice. MaSC-enriched basal cell expansion and increased self-renewal and in vivo regenerative capacity of MaSCs are observed in the mammary glands of MMTV-Id1 transgenic mice. Furthermore, MMTV-Id1 mice develop ductal hyperplasia and mammary tumors with highly expressed basal markers. Id1 also increases breast cancer stem cell (CSC) population and activity in human breast cancer lines. Moreover, the effects of Id1 on normal and malignant stem cell activities are mediated by the Wnt/c-Myc pathway. Collectively, these findings provide in vivo genetic evidence of Id1 functions as an oncogene in breast cancer and indicate that Id1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to breast tumor development.

MEL-18 loss mediates estrogen receptor-α downregulation and hormone independence.

The polycomb protein MEL-18 has been proposed as a tumor suppressor in breast cancer; however, its functional relevance to the hormonal regulation of breast cancer remains unknown. Here, we demonstrated that MEL-18 loss contributes to the hormone-independent phenotype of breast cancer by modulating hormone receptor expression. In multiple breast cancer cohorts, MEL-18 was markedly downregulated in triple-negative breast cancer (TNBC). MEL-18 expression positively correlated with the expression of luminal markers, including estrogen receptor-α (ER-α, encoded by ESR1). MEL-18 loss was also associated with poor response to antihormonal therapy in ER-α-positive breast cancer. Furthermore, whereas MEL-18 loss in luminal breast cancer cells resulted in the downregulation of expression and activity of ER-α and the progesterone receptor (PR), MEL-18 overexpression restored ER-α expression in TNBC. Consistently, in vivo xenograft experiments demonstrated that MEL-18 loss induces estrogen-independent growth and tamoxifen resistance in luminal breast cancer, and that MEL-18 overexpression confers tamoxifen sensitivity in TNBC. MEL-18 suppressed SUMOylation of the ESR1 transactivators p53 and SP1, thereby driving ESR1 transcription. MEL-18 facilitated the deSUMOylation process by inhibiting BMI-1/RING1B-mediated ubiquitin-proteasomal degradation of SUMO1/sentrin-specific protease 1 (SENP1). These findings demonstrate that MEL-18 is a SUMO-dependent regulator of hormone receptors and suggest MEL-18 expression as a marker for determining the antihormonal therapy response in patients with breast cancer.

Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.

Loss of Mel-18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway.

Mel-18 has been proposed as a negative regulator of Bmi-1, a cancer stem cell (CSC) marker, but it is still unclear whether Mel-18 is involved in CSC regulation. Here, we examined the effect of Mel-18 on the stemness of human breast CSCs. In Mel-18 small hairpin RNA (shRNA)-transduced MCF-7 cells, side population (SP) cells and breast CSC surface marker (CD44(+)/CD24(-)/ESA(+))-expressing cells, which imply a CSC population, were enriched. Moreover, the self-renewal of CSCs was enhanced by Mel-18 knockdown, as measured by the ability for tumorsphere formation in vitro and tumor-initiating capacity in vivo. Similarly, Mel-18 overexpression inhibited the number and self-renewal activity of breast CSCs in SK-BR-3 cells. Furthermore, our data showed that Mel-18 blockade up-regulated the expression of the Wnt/TCF target Jagged-1, a Notch ligand, and consequently activated the Notch pathway. Pharmacologic inhibition of the Notch and Wnt pathways abrogated Mel-18 knockdown-mediated tumorsphere formation ability. Taken together, our findings suggest that Mel-18 is a novel negative regulator of breast CSCs that inhibits the stem cell population and in vitro and in vivo self-renewal through the inactivation of Wnt-mediated Notch signaling.

The in vitro and in vivo anti-tumor effect of KO-202125, a sauristolactam derivative, as a novel epidermal growth factor receptor inhibitor in human breast cancer.

Epidermal growth factor receptor (EGFR) is one of the most promising targets for cancer therapy. Here, we show the in vitro and in vivo anticancer effects and associated mechanisms of KO-202125, one of the synthesized aristolactam analogs, as a novel EGFR inhibitor, in EGFR-overexpressing cancer cell lines. KO-202125 showed more effective growth inhibition and apoptosis induction than gefitinib, a representative EGFR inhibitor, in various EGFR-overexpressing human cancers including estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. Epidermal growth factor receptor phosphorylation at Tyr1068 was reduced and, consequently, the association of EGFR with p85 was decreased by KO-202125 treatment in MDA-MB-231 cell lines. This led to inactivation of the PI3K/Akt pathway, and consequently suppression of activation of the Wnt pathway and enhancement of the nuclear import of p27Kip1. KO-202125 treatment in nude mice injected with MDA-MB-231 cells showed inhibition of tumor growth without toxicity. Collectively, our results showed the possibility of KO-202125 as an effective therapy agent of EGFR-overexpressing cancer cells through reduced EGFR activity and downregulation of the Akt pathway.

Constitutive overexpression of Id-1 in mammary glands of transgenic mice results in precocious and increased formation of terminal end buds, enhanced alveologenesis, delayed involution.

Inhibitor of differentiation-1 (Id-1) has been shown to play an essential role in cell proliferation, invasion, migration, and anti-apoptosis. However, the effect of Id-1 in mammary gland development remains unknown. Here, we generated MMTV-Id-1 transgenic mice to study the role of Id-1 in mammary gland development. In virgin mice, Id-1 overexpression led to precocious development and delayed regression of terminal end buds (TEBs) compared with wild-type mice. The number of BrdU-positive cells and the expression of Wnt signaling molecules, ß-catenin and cyclin D1, which regulate ductal extension and TEB formation in virgin, were statistically higher in Id-1 transgenic mice than in wild-type mice. Id-1 also had an effect on the formation and proliferation of lobuloalveolar structures during early and mid-pregnancy. Id-1 transgenic mice had more lobulated and prominent alveolar budding than wild-type mice and had significantly greater counts of lobuloalveolar structures in early pregnancy. The expression of BrdU, ß-catenin, and cyclin D1 was also predominantly increased in Id-1 transgenic mice. Moreover, Id-1 transgenic mice showed delayed involution. Id-1 regulated the expression levels of anti-apoptotic Bcl-2 and pro-apoptotic Bax, and resulted in delay of apoptotic peak during postlactational involution. We also found that Id-1 was able to modulate expression of the regulators of Wnt/ß-catenin signaling such as phospho-Akt, BMP2, FGF3, and RAR-ß in tubuloalveolar development of mammary glands. Taken together, our results suggest that Id-1 plays a pivotal role in mammary gland development through Wnt signaling-mediated acceleration of precocity and alveologenesis and Bcl-2 family members-mediated delay of involution.

Mel-18 negatively regulates INK4a/ARF-independent cell cycle progression via Akt inactivation in breast cancer.

Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

Anticancer effects of wogonin in both estrogen receptor-positive and -negative human breast cancer cell lines in vitro and in nude mice xenografts.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)-positive and -negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50-200 microM), independently of its ER status, in a time- and concentration-dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK-3beta and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt-mediated canonical Wnt signaling pathway. ER expression was downregulated in ER-positive cells, while c-ErbB2 expression and its activity were suppressed in ER-negative SK-BR-3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA-MB-231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ER-positive and -negative breast cancers, particularly against the more aggressive and hormonal therapy-resistant ER-negative types.

The antitumor effect of LJ-529, a novel agonist to A3 adenosine receptor, in both estrogen receptor-positive and estrogen receptor-negative human breast cancers.

Agonists to A3 adenosine receptor (A3AR) have been reported to inhibit cell growth and/or induce apoptosis in various tumors. We tested the effect of a novel A3AR agonist generically known as LJ-529 in breast cancer cells. Anchorage-dependent cell growth and in vivo tumor growth were attenuated by LJ-529, independently of its estrogen receptor (ER) alpha status. In addition, apoptosis was induced as evidenced by the activation of caspase-3 and c-poly(ADP)ribose polymerase. Furthermore, the Wnt signaling pathway was down-regulated and p27(kip) was induced by LJ-529. In ER-positive cells, the expression of ER was down-regulated by LJ-529, which might have additionally contributed to attenuated cell proliferation. In ER-negative, c-ErbB2-overexpressing SK-BR-3 cells, the expression of c-ErbB2 and its downstream extracellular signal-regulated kinase pathway were down-regulated by LJ-529. However, such effect of LJ-529 acted independently of its receptor because no A3AR was detected by reverse transcription-PCR in all four cell lines tested. In conclusion, our novel findings open the possibility of LJ-529 as an effective therapeutic agent against both ER-positive and ER-negative breast cancers, particularly against the more aggressive ER-negative, c-ErbB2-overexpressing types.